血管形成素-1基因转染对血管内皮细胞影响的实验研究

Effect of angiopoietin-1 gene transfer mediated by recombinant adenovirus on endothelial cell

目的 探讨血管形成素 1(Ang1)基因重组腺病毒 (Ad .Ang1)的获取及其对人脐静脉上皮细胞 (ECV30 4 )的影响。方法 以逆转录聚合酶链式反应 (RT PCR)自人脾组织中克隆出Ang1全长cDNA ,测序后克隆到Ad5腺病毒载体中 ,制备纯化的Ad .Ang1;以Ad .Ang1转染ECV30 4细胞 ,1至 7d后 ,用RT PCR与Westernblot分别检测ECV30 4中Ang1转录与表达情况 ,同时观测其对ECV30 4的影响。结果 克隆的Ang1DNA片段为 15 15bp的全长cDNA。所获得的Ad .Ang1与 β 半乳糖苷酶 (LacZ)基因重组腺病毒 (Ad .LacZ)滴度分别为 5 6× 10 8pfu/ml、5 3× 10 8pfu/ml。β 半乳糖苷 (X gal)染色显示LacZ基因转染Hela细胞 ,蓝染率可达 95 %以上。Ang1基因转染后第 1、3、7d可检出mRNA表达 ,第 3、7d可检测到其蛋白表达。XTT检测显示Ad .Ang1组与对照组相比 ,各时间点的OD值差异不显著 ,P >0 0 5。结论 Ad .Ang1在体外培养的ECV30 4细胞中能有效表达 ,但对ECV30

Objective To generate the angiopoietin-1 recombinant adenovirus and study the potential effects of angiopoietin-1 via adenovirus mediated gene transfer on endothelial cell.Method Angl cDNA was obtained by RT-PCR from human spleen and was inserted into E1,E3 substituted adenovirus vectors pAxCAwt following the co-transfection of 293 host cell line.The viral stocks were demonstrated to be free of replication-competent wild type adenoviruses.We detected the transcription and expression of Angl gene in ECV304 cells by RT-PCR and western blot respectively,observed the effects of Ad.Angl on the proliferation of endothelial cell.Results The results showed that Angl cDNA cloned in our laboratory was 1515bp in length.The viral titers of Ad.Angl and Ad.LacZ were 5.6×10 8pfu/ml,5.3×10 8pfu/ml respectively.X-gal staining of hela cells infected with Ad.LacZ showed 95% blue staining at 100 of MOI value.RT-PCR revealed that the Angl gene could be transcripted in transfected ECV304 cells. The expression of Angl by western blot could be found in the ECV304 cells at days 3 and 7;XTT test indicated that the Ad.Angl had no promotive effects on the proliferation of endothelial cell.Conclusion Ad.Angl in our laboratory could be expressed efficiently in ECV304 cells,but it had no promotive effects on the proliferation of endothelial cell.